Galectin-3 (Gal-3) is a beta-galactoside binding lectin displaying both intracellular and extracellular immune functions. In Schistosoma mansoni infection, Gal-3 has been associated with the induction of a T helper 2 response. Whereas dendritic cells (DCs) play a pivotal role in the regulation of T cell differentiation, little is known about the regulation of Gal-3 expression in DCs. In this study we determined Gal-3 mRNA and protein levels during in vitro differentiation of human monocytes into immature DCs (iDCs), by culturing monocytes in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Gal-3 mRNA levels show a moderate, transient increase during iDC generation, accompanied by elevated cell-associated Gal-3 protein. Our data show that culturing monocytes with IL-4 alone strongly increases Gal-3 mRNA levels, whereas GM-CSF induces a low increase in Gal-3 mRNA. The combined data indicate that GM-CSF reduces IL-4 induced Gal-3 mRNA levels during the generation of iDC. Remarkably, stimulation of monocytes with GM-CSF results in secretion of significant amounts of Gal-3 in the medium, whereas iDCs do not release detectable amounts of Gal-3, indicating a suppressive role of IL-4 on GM-CSF induced Gal-3 secretion. Finally, our data demonstrate that all differentiated cell types tested show a significantly lower capacity to bind Gal-3 on the cell surface than monocytes. In conclusion, Gal-3 expression in iDCs is restricted, and Gal-3 protein is localized mainly intracellular, due to the opposite actions of IL-4 and GM-CSF. By these properties, the DCs may be protected against Gal-3 induced phosphatidylserine (PS) exposure and/or apoptosis.